Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular β sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo–electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its β region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Our findings indicate that the purified GFP-ΦCb5 (titer of ~5 x 10 9 pfu ml -1 ) has a one-log drop in titer compared to the mixture (titer of ~8 x 10 10 pfu ml -1 ).Recognizing that the purification process may result in some loss of GFP-ΦCb5, we estimate that the original particle numbers of WT-ΦCb5 and GFP-ΦCb5 are on the same order in our two-plasmid system.In addition, the plasmid for GFP-Mat (pBAD33) is of a lower copy number compared to the plasmid of WT-ΦCb5 (pET28+) in our two-plasmid system.Given the comparable amounts of GFP-ΦCb5 with WT-ΦCb5, we estimate the incorporation efficiency of the GFP-Mat into infectious phage particles should be at least not worse than the WT-Mat.

Fig. S2 .
Fig. S2.Cryo-EM data processing workflow of the Tad pilus.(A) Cryo-EM data processing diagram of the Tad pilus.(B) A representative view of the micrograph after motion correction.The white scale bar denotes 500 Å. (C) Selected 2D class averages of the Tad pilus.The white scale bar denotes 100 Å. (D) Local resolution estimations of the cryo-EM density map of the Tad pilus showing both surface (left) and cut-open (right) views.(E) The gold-standard Fourier Shell Correlation (FSC) curves of the density map of the Tad pilus.

Fig. S3 .
Fig. S3.Hydrophobic interaction and hydrogen bonds within the Tad pilus.(A) A schematic diagram showing one pilin and six neighboring pilins.(B) The same interacting pilins, denoted in panel A, are colored on the pilus model.(C-H) Close-up views, showing the residues responsible for the hydrophobic interaction between neighboring pilin pairs.(I-J) Hydrophobicity analysis of the Tad pilus in the surface view (I) and cut-open view (J).(K) A close-up view of the red box in (J) showing hydrogen bonds (dashed blue lines) network of Glu19-Ala17 and Glu19-Tyr20 in the core of pilus.

Fig. S4 .
Fig. S4.Cryo-EM data processing workflow of ΦCb5 virion and ΦCb5-Tad complex.(A) A diagram showing the cryo-EM data processing workflow.Local resolution of the Virion, Mat, and Mat-pilus maps are color coded.(B) A representative cryo-EM micrograph ΦCb5 mixed with Tad pili.The white scale bar denotes 500 Å. (C) Gold-standard Fourier Shell Correlation plot showing overall resolutions for the Virion map, Mat local map and Mat-pilus local map, respectively.

Fig. S5 .
Fig. S5.Unique characteristics of the ΦCb5 Coat shell and the calcium ions in the ΦCb5 capsid.(A) The ribbon model depicts adjacent pentamer and hexamer of coat proteins of ΦCb5 (top row), MS2 (middle row) and Qβ (bottom row), respectively.The diameter of the opening at the center of hexamer is labeled.(B-C) Zoom-in views of the density and model in the black (B) and red (C) boxed regions on the ΦCb5 Coat shell depicted in (A).Negatively charged amino acid sidechains interact with calcium ions.

Fig. S6 .
Fig. S6.Interaction of the capsid proteins with the RNA four-way junction domain at the 3' end of the gRNA.(A) The overall interaction 3' end domain (colored density) interacts with Mat (pink model) and Coat dimers (blue models).(B) Cryo-EM density of the RNA domain matches a four-way junction model of RNA residues 3652-3762.The three long RNA stem-loops are labeled R1, V and U1, respectively.(C-D) Interaction between the Coat dimer with Stem-loops V (C) and R1 (D), respectively.Hydrogen bonds and salt bridge are labeled with blue and black dashed lines, respectively.(E) Electrostatic surface potential of the Mat showing the U1 binding region (salmon circle) is highly positively charged.

Fig. S7 .
Fig. S7.Adsorption of ΦCb5 with C. crescentus subspecies and mutants.(A) Sequence comparison of pilins between of NA1000 and bNY30a strains.(B) A scheme showing the procedure of the adsorption assay.Briefly, ΦCb5 are incubated with C. crescentus subspecies and mutants, allowing pilus binding to occur.The sample is then centrifuged to collect the unbound ΦCb5 phages in the supernatant to further infect the host strain bNY30a.The efficiency of plating (EOP) of the unbound ΦCb5 to bNY30a then be measured.Schematic illustration was created with BioRender.(C) EOP of the unbound ΦCb5 showing ΦCb5 binds poorly to NA1000strain as to the Tad major pilin knock-out strain, NA1000 ΔpilA.However, ΦCb5 binds to a pilin-modified strain, bNY30a pilAT36C, as good as the host strain bNY30a.In the top right inset, the sites for the cysteine mutation for the amino acid T36 of the pilins are labeled as gold spheres on the pilus model and reside outside the Mat-pilus interface.The T36C mutation allows maleimide dye labeling for the fluorescence imaging.(D) Model fitting in map to show the match of pilin models in our cryo-EM density.

Fig. S8 .
Fig. S8.Purification, fluorescent labeling, and infectivity test of GFP-ΦCb5.(A) The anti-GFP nanobody used in the affinity purification of the GFP-ΦCb5.(B) Fluorescence imaging of GFP-ΦCb5 binding to bNY30a cells.The white scale bar denotes 10 μm.(C) The titer result of the lysate containing the mixture of WT-ΦCb5 and GFP-ΦCb5 was compared with that of purified GFP-ΦCb5.Numbers indicate the dilution of the phage sample by a multiple of ten from left to right.Our findings indicate that the purified GFP-ΦCb5 (titer of ~5 x 10 9 pfu ml -1 ) has a one-log drop in titer compared to the mixture (titer of ~8 x 10 10 pfu ml -1 ).Recognizing that the purification process may result in some loss of GFP-ΦCb5, we estimate that the original particle numbers of WT-ΦCb5 and GFP-ΦCb5 are on the same order in our two-plasmid system.In addition, the plasmid for GFP-Mat (pBAD33) is of a lower copy number compared to the plasmid of WT-ΦCb5 (pET28+) in our two-plasmid system.Given the comparable amounts of GFP-ΦCb5 with WT-ΦCb5, we estimate the incorporation efficiency of the GFP-Mat into infectious phage particles should be at least not worse than the WT-Mat.

Fig. S9 .
Fig. S9.The modified two-plasmid system shows infectivity and fluorescent binding of GFP-ΦCb5 to Tad pili of C. crescentus.(A) The modified two-plasmid system introduces two premature stop codons to inhibit the mat gene on the WT-ΦCb5 genome.The stop signs annotate the locations of the two premature stop codons: one replaces the start codon while the other is inserted 540 bp downstream the start codon in the middle of the mat gene.(B) As this new version of GFP-ΦCb5 contains phage RNA with the mat gene knocked out, it can still bind the host, deliver the RNA and lyse the cell, but not able to produce new infectious virions inside the cell for subsequent infection of new cells as in a standard titer assay.Therefore, we have performed the following assay with proper controls.The E. coli cells with our two-plasmids, encoding the GFP-Mat (pBAD33) and Mat-knockout ΦCb5 genome (pET28+), were induced differently (from top to bottom in Panel B): by both Arabinose (Ara) and IPTG, by IPTG only, by Ara only, uninduced.These E. coli cells were then washed and lysed within PYE media, with the lysate applied to C. crescentus plate lawns.We observed only the lysate with both plasmids induced, containing these modified GFP-ΦCb5 particles, shows clearing of the C. crescentus lawn.Numbers indicate the log dilutions of the sample by a multiple of ten from left to right.Bottom row is another control by applying the PYE media alone onto the C. crescentus lawn, which showed no C. crescentus killing.(C) Fluorescence imaging of modified GFP-ΦCb5 binding to C. crescentus bNY30a cells.White arrowheads point to representative Tad pili from the cells.The white scale bar denotes 5 μm.